Use of chitinase and chitosanase to form sphaeroplasts from Pilobolus sporangiospores

Extracting cell contents, such as DNA and protein, from cells with cell walls creates a special challenge because the wall has to be removed without damaging the contents of the cell itself. One method to remove cell walls is to use enzymes to break down cell wall components to release the cell's sphaeroplast. This study was undertaken to develop a technique that could provide sphaeroplasts from sporangiospores of the zygomycete, Pilobolus. Sphaeroplasts from some fungi have been prepared by digesting the germ tube cell wall more successfully than digesting the spore wall itself. However, sphaeroplasts were released more readily from ungerminated Pilobolus sporangiospores than from the germ tubes growing from spores. An enzyme suspension containing a combination of chitinase and chitosanase readily produced intact sphaeroplasts from which sufficient DNA could be extracted to do sequencing studies.